The present invention relates generally to processes having an antimicrobial objective. More specifically, it relates to protozoicides, that is, processes for the killing of protozoa.
The hop plant, Humulus lupulus, is a twining vine that is a member of the mulberry (Cannabinaceae) family. It has inconspicuous flowers, the pistillate ones of which form glandular, cone-shaped catkins which, when ripe and dried, find familiar use to impart a bitter flavor to beverages derived from malt. Hop has been used to flavor and preserve wort and beer since the 12th century in Germany and the 15th century in England. Its resins, which reside in the yellow glands of its cones, have been known to possess antimicrobial properties. Those resins are extracted from the cones, usually by supercritical carbon dioxide treatment, or by extraction with organic solvents, and are today most often used in the form of extracts.
With respect to the chemical nature of hop compounds, they are weak acids classified as alpha bitter resins and beta bitter resins or bitter acids. Alpha bitter acids are represented by humulone and its cogeners, cohumulone, adhumulone, prehumulone and posthumulone. The beta resins are represented by lupulone, and its cogeners, colupulone, adlupulone, and prelupulone. The alpha and beta acids have alicyclic structures (2,4-cyclohexodine-1-one), but their cogeners differ in the nature of the acyl side chain. While the alpha acids and their derivatives, the iso acids, contribute most to the bitterness of beer, there are many other, sometimes minor constituents that contribute to preservation and bitterness. One of these compounds is xanthohumol, which has also been found to fall within the scope of the present invention.
The antimicrobial properties of hop compounds are well-known; their extracts primarily are used to inhibit the growth of gram positive microbes, and they are usually inactive against gram negative bacteria. Yeast is not inhibited by hop compounds. Fungi are either not inhibited or inhibited at unfavorably high concentrations of hop extracts. Prior to our work, little if anything was known about the effect of hop extracts on protozoa, the only possible exception being work in U.S. Pat. No. 5,827,895 by Nutter et al. about the inhibition of the pathogenic protozoa Leishmania by a chemically modified hop derivative, hexahydrolupulone. However, it has now been found that different hop extracts have a decidedly positive effect in killing a broad range of protozoa.
There are many pathogenic protozoa that lead to human diseases. Among these are Entameba histolytica, Cryptosporidium parvum, Giardia lamblia, and others that are pathogenic in the intestinal tract. Campers often have to purify water and remove pathogenic protozoa to make it potable. Still other protozoa are systemic, such as plasmodium, the causative agent of malaria, and trypanosomes.
It is, therefore, a primary object of the present invention to formulate a-process or method for killing protozoa by the use of compositions that are harmless to humans. As will be apparent, the objective of this work has been to achieve that goal.
The present invention is in the form of a method for killing protozoa. The agent for accomplishing this purpose is an effective amount of extract from the hop plant, which extract is composed of alpha resins, beta resins, xanthohumol and combinations thereof.
When used herein, the term, alpha-resins, is intended include alpha resins per se, e.g., humulone, and also its derivatives, such as iso alpha resin and tetra iso alpha resin. When alpha resin as so defined is the agent for killing protozoa, it is preferably in the form of the tetra iso alpha resin, which is water-soluble. In this instance the minimum effective amounts of the alpha resin are about 0.2 to about 20 ug/ml. when the protozoa are ciliates or flagellates. When the protozoa are in the form of amoebae, the-minimum effective amount varies from about 100 to about 500 ug/ml., as amoebae appear more resistant to alpha resin than ciliates or flagellates.
When beta resins are utilized with ciliates or flagellates, the effective amount is about 0.1 to about 2.0 ug/ml. Xanthohumol appears to be more lethal to such protozoa than beta resin, the quantity necessary to achieve a 100% kill rate varies from about 0.05 to 1.0 ug/ml.
It has also been discovered that a greatly improved, synergistic effect is obtained when carbon dioxide is used in combination with hop resins against protozoa. Thus, when the extract solution of hop resins is subjected to treatment with carbon dioxide, such as by bubbling carbon dioxide gas through the extract solution prior to subjecting the protozoa to treatment with the solution, a superior kill of protozoa is obtained. For example, when the alpha resin as herein defined is used against ciliates and flagellates, prior treatment of the extract solution with carbon dioxide results in killing the protozoa at a concentration of only 0.05 to 0.1 ug of extract per ml. of solution. The same is true for solutions of beta resins. Amoebae are killed with a concentration of 10 to 100 ug/ml. of hop resin in a solution that has been pre-treated with carbon dioxide.
In still another aspect of the present invention, it has also been discovered that solutions which have been carbonated but without hop extract have a lethal effect on protozoa. Thus, as set forth in the detailed description of one preferred embodiment of our invention, when carbon dioxide was bubbled through an aqueous medium for 30 seconds prior to inoculation of the protozoa without the addition of hop extract, death occurred within about 40 to 50 minutes.
These and other features and advantages of the present invention will be more apparent when considered in connection with the following, detailed description of certain preferred embodiments of our invention.
The hop resins used in this work were obtained from S. S. Steiner, Inc. of New York, N.Y. Those resins were the alpha resin in the form of a 30% aqueous solution of its iso alpha resin derivative, and tetra iso alpha resin derivative in a 19% aqueous solution. Beta resin, i.e., lupulone, was obtained in pure form, as was xanthohumol (99.3% pure).
With regard to the protozoa used in our tests, in Table 1 the protozoa are listed, as well as the medium from which they were obtained, the temperature and period of incubation. As stated in Table 1, all of the protozoa were obtained from Carolina Biological Supply Co., of Burlington, N.C. Six different protozoa are listed in Table 1. They were used in the examples of protozoicidal activity which follow.